Pancreatic ductal adenocarcinoma (PDAC) is rapidly becoming the second leading cause of cancer-related deaths in the U.S. The genetic landscape of PDAC shows prevalent mutations of KRAS; however, expression of mutant KRAS (KRASmt) alone at the adult stage is insufficient to drive PDAC, suggesting that a second hit is required. KRASmt was previously regarded as an oncogene and thought to be fully active, yet recent studies have shown that an endogenous level of KRASmt is not fully active. Rather, it can be hyperactivated by pancreatic cancer risk factors, including pancreatitis and obesogenic high-fat diet challenge, which act as the second hit to promote PDAC with high penetrance. However, the molecular mediator linking these risk factors to KRASmt hyperactivation remains elusive. NADPH oxidases (NOX) are major enzymes activated by KRASmt for the generation of reactive oxygen species and oxidative stress in cancer. Notably, our preliminary data have shown that inhibition of NOX suppresses KRASmt activation, indicating that NOX is not only a downstream effector but also a potential upstream regulator of KRASmt. Based on these observations, we hypothesize that pancreatic cancer risk factors, including pancreatitis and chronic high-fat diet consumption, facilitate the formation of a sustained NOX and KRASmt co-activation partnership, which leads to full-blown PDAC. Targeted inhibition of NOX breaks the partnership promoted by these risk factors, thus hampering pancreatic tumorigenesis. To test this hypothesis, we will employ novel inducible genetically engineered mouse models expressing endogenous levels of KRASG12D with ablation of NOX docking subunit p22phox in pancreatic acinar cells and expose the mice to inflammatory stimuli or obesogenic high-fat diet. Similarly, mutant p53 also facilitates the NOX-KRASG12D co- activation partnership to promote aggressive PDAC. We will ablate p22phox in pancreatic acinar cells of the mice expressing both KrasG12D/+ and p53R172H/+. The objective of this proposal is to determine if NOX is the bona fide critical molecular mediator linking these pancreatic cancer risk factors to KRASmt hyperactivation, which drives pancreatic neoplastic progression, a fundamental unanswered question in the pancreatic cancer field. If proven, this study will delineate the molecular underpinnings and cellular events of the synergistic cooperation among oncogenic KRAS, NOX, and pancreatic cancer risk factors, and will provide insights into novel preventive and therapeutic strategies against this devastating disease in humans.